This invention relates to new compositions of heparins of low molecular weight (HLMW) constisting of a limited number of heparin fragments that have a 4-enopyranosyl uronate group at its non-reducing end.
Herapin is a mucopolysaccharide sulphate of animal origin, extracted from mammal intestine or lung (cow, lamb, pig) and used for some time now in human therapy for prevention and treatment of thromboembolic diseases. It is well known that the use of heparin is accompanied by very upsetting haemorrhaging effects and its daily administration, three subcutaneous or intravenous injections, constitutes a very considerable drawback.
During the course of the last few years different chemical methods have been used for depolymerising heparin, such as:
treatment with sodium nitrite in acid medium
alkali treatment of esters
use of free radicals generated in the presence of oxygenated water
treatment of an quaternary ammonium salt of heparin in non-aqueous medium with
a strong base according to a beta-elimination mechanism.
These methods allow variable yields to be obtained, mixtures of fragments of heparin in which the average molecular weight and the anti-coagulant activity vary according to the procedure and the conditions of the operation. The heparins of low molecular weight (HLMW) described in the state of the art or commercialised are obtained according to different depolymerisation procedures. Their average molecular weight (Mw) lies between 4,000 and 6,000 Daltons.
Currently it is recognised that the anti-thrombic activity of HLMW""s is due principally to their capacity for activating anti-thrombin III, a plasma protein and potent inhibition of activated factor X and thrombin. In this way it is possible to measure the anti-thrombic activity of heparin by means of tests specific to the inhibition of these factors.
The research carried out by different authors in recent years shows that fragments of oligosaccharides of heparin consisting of short chains of medium molecular weight  less than 4,800 Daltons have a selective action on activated factor X and have little effect on the global coagulation measured using methods of the farmacopea. It has been found that if fragments of very low molecular weight are required that have a strong anti Xa activity, it is preferable to use a selective depolymerisation technique in non-aqueous medium, as is described in the patent U.S. Pat. No. 4,981,955 that does not run the risk of attacking the site of binding to anti-thrombin III.
In view of the background and state of the art described above, the present application has developed, using a procedure in non-aqueous medium, the controlled depolymerisation of heparin that allows a new family of HLMW to be obtained rich in oligosaccharides of low molecular weight that have a high anti-Xa activity and a low anti IIa activity, and that can be represented by the general formula: 
in which:
n can vary between 1 and 12
R1=H or SO3Na
R2=SO3Na or COCH3 
Said heparin of very low molecular weight is obtained by selectively depolymerising the heparin in non-aqueous medium according to a procedure of beta elimination.
The compositions of heparins of very low molecular weight according to this invention are characterised by a molecular weight lying between 2,000 and 4,000 Daltons, an anti factor Xa activity of at least equal to 100 I.U./mg and contain a strong proportion, up to 75%, of oligosaccharides of low degree of polymerisation that go from hexasaccharide (n=1) to dodecasaccharide (n=4). These compositions are useful in the prophylaxis and treatment of venous and arterial thrombosis. They can be used as anti-thrombic medication.
The HLMW known and exploited commercially contain small proportions of oligosaccharides of low molecular mass, notably the oligosaccharides whose degree of polymerisation goes from hexasaccharide to dodecasaccharide.
The compositions of heparin, fruit of the present invention, have as a main characteristic, the fact that they contain a strong proportion, up to 75%, of such oligosaccharides. Furthermore, these oligosaccharides have a high anti Xa activity ( greater than 100 I.U./mg) giving them a long lasting and high anti-thrombic activity. Said compositions of heparin have an anti-Xa activity lying between 100 and 150 I.U./mg and whose anti-factor Ia activity is less than or equal to 10 I.U./mg.
The average molecular weight of the compositions of heparin of the present application lie between 2,000 and 4,000 Daltons and because:
they contain from 25 to 60% of oligosaccharides of molecular weight less than 2,000 Daltons;
they contain from 40 to 75% of oligosaccharides of molecular weight lying between 2,000 and 6,000 Daltons; and
they contain less than 15% of oligosaccharides of molecular weight greater than 6,000 Daltons.
The heparin compositions of the present invention are composed of mixtures of oligosaccharides or fragments of heparin. The percentage of fragments that form part of the present invention are as follows:
contain less than 10% of fragments in which n lies between 10 and 12;
contain from 80 to 90% of fragments in which n lies between 1 and 6; and
contain less than 15% of the fragments in which n lies between 7 and 9.
The present invention is illustrated by the following examples, without these examples limiting the scope of the invention.
The molecular weight (Mw), the molecular distribution, as well as the anti-factor Xa activities have been determined according to techniques described in the monograph no. 828 xe2x80x9cHeparin of low molecular weightxe2x80x9d of the Third Edition of the Pharmacopea Europea.